MiRNA have the potential to revolutionize both cancer detection and therapeutics as they exert control over nearly every cellular process from differentiation to growth and senescence. Although over 2000 miRNA are predicted to exist, studies have suggested that smaller subsets of only 5-50 miRNA are often the critical players in any specific disease or cellular process. However, existing microarray and qRT-PCR techniques are too slow and too expensive to screen large numbers of samples across these targeted miRNA panels, a critical task in the validation of miRNA cancer tests. In Phase I, we developed a multiplexed ligation assay to analyze 10-100 miRNA in a single reaction called Ligo-miR. We demonstrated that Ligo-miR expression profiles had high correlation to the gold standard Taqman qRT-PCR assays (R2=0.89-98). Yet, Ligo-miR required 10-20x less hands-on time, had 5x less cost, and had 20x higher overall sample-throughput, successfully achieving the goal of fast and low-cost, targeted miRNA profiling. Ligo-miR incorporates 2 sequential ligation steps to generate miRNA specific reaction products encoded by length. This flexible, length-based multiplexing is specifically designed to enable detection using an array of DNA sizing methods including polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), and single molecule free solution hydrodynamic separation (SML-FSHS) such that sensitivity, multiplex capability, and sample throughput can be tailored a wide spectrum of miRNA applications and user requirements. In this Phase II SBIR proposal, we will leverage the Phase I results to develop a platform of commercial products based on Ligo-miR technology for broad research applications. First, Ligo-miR EZ will be developed for general profiling of cells and tissues using fluorescent PAGE detection to achieve a sensitivity of 20 copies per cell. Nearly all molecular labs will have a thermal cycler and PAGE apparatus, making fast and low cost multiplex analysis available to all. Next, Ligo- miR CE will be developed for applications requiring the highest sensitivity and throughput such as single cell analysis or clinical analysis of blood. It incorporates automated CE detection to increase sample throughput to thousands of samples per day and a post-amplification to boost sensitivity to single cell levels. Then, Ligo-miR HD will be developed for challenging applications using limited samples or that could benefit from lab-on-a-chip integration such as FFPE analysis and single cell. It uses microfluidic, single molecule technology for high sensitivity detection using minute amounts of sample. Finally, we will perform a technical validation and benchmark each Ligo-miR assay against Taqman qRT-PCR.